PROJECT SUMMARY/ABSTRACT To support the cellular demands of unchecked proliferation and to maintain a malignant cell state through successive rounds of cellular division, cancer cells depend on lineage-specific, dysregulated gene expression programs. Thus, the chromatin regulators that underlie chromatin-dependent transcription control have been considered attractive targets to disrupt or diminish pathogenic transcriptional signaling. Previously, I led a research effort that established ENL, a transcriptional co-activator and chromatin reader protein, as a critical requirement for the survival of acute leukemia in cellular and animal model systems. Notably, its YEATS domain, a crotonyl-lysine-binding chromatin reader domain that anchors ENL at the promoters of transcriptionally active genes, is required for the pro-proliferative effects of ENL in leukemia. Moreover, preliminary data suggests that KAT6A, a histone acetyltransferase and crotonyl-lysine chromatin reader protein, is also required for acute leukemia pathogenesis. Here, I propose to further consider crotonyl-lysine reader proteins as potential therapeutic opportunities in acute leukemia with the following three specific aims. In the first aim, I will determine the cellular pathways and co-factors regulating response and resistance to loss of ENL in acute leukemia. Genetic experiments proposed within this aim will determine the cellular pathways and co-factors regulating ENL target biology to understand its mechanism of action in promoting leukemia cell proliferation and survival. In the second aim, I will test the hypothesis that the ENL YEATS domain can be disrupted by small-molecule inhibitors, which will serve both as chemical probes of ENL protein function and as starting points for translational development of ENL-targeted leukemia therapy. I will apply high-throughput chemical screens, structure-based drug design, and iterative medicinal chemistry to discover and optimize first-in-class ENL YEATS inhibitors. In the third aim, I will assess the role of crotonyl-lysine recognition by KAT6A in leukemia pathogenesis; I will use genetic approaches and targeted protein degradation to determine whether crotonyl-lysine recognition by the double PHD finger domain (DPF) of KAT6A is required for acute leukemia survival. Each proposed aim operates within early-stage drug discovery efforts (i.e. target validation and chemical tool discovery) and if successful, will provide biological insights and chemical tools that will enable timely translational development of novel, molecularly-targeted cancer medicines. In keeping with the model of open-innovation in academic drug discovery, all chemical probes, biological tools, and genomic datasets derived from this work will be made publicly and freely available, without restriction on use, so to broaden and accelerate the impact of this work.